Abstract The divergence of the somatotrope and lactotrope lineages in the anterior pituitary constitutes a highly informative, medically relevant, and well-characterized model of mammalian cellular differentiation. Remarkably, the differentiation of both lineages is dependent on the activity of the same pituitary-specific transcription factor, Pit-1 (POU1-F1). Landmark genes defining each of the two lineages, such as Growth Hormone (GH) and Prolactin (Prl), are under direct control of Pit-1 dependent cis-regulatory elements. Loss of Pit-1 expression results in absence of both cell types from the anterior pituitary in mice and humans with consequent combined hormone deficiency syndromes. Despite the central importance of Pit-1 to development and function of the anterior pituitary, the mechanisms and pathways that it activates to drive the differentiation and maintenance of the somatotrope and lactotrope lineages remain unclear. We hypothesize that Pit-1, acting in conjunction with differentially expressed transcription factors, controls the divergence of the somatotrope and lactotrope lineages by binding to lineage-defined cis-regulatory elements, mediating long- range transcriptional interactions, organizing nucleosomal architectures, and defining lineage-dependent three dimensional (3D) chromatin networks. In Aim 1, we test the hypothesis that Pit-1 occupancy at the major enhancer element within the human GH locus control region (HSI) activates a series of temporally-defined functions that are critical to the robust and selective activation of hGH-N transcription in the pituitary somatotrope and its reciprocal repression in the lactotrope lineage. In Aim 2 we test the hypothesis that the ability of Pit-1 to drive lineage divergence depends on its interactions with cooperating transcriptional factor(s). Candidate factors are identified by comparative transcriptome analyses of primary flow-sorted somatotropes and lactotropes and are validated by a set of compelling functional assays. In Aim 3 we test the hypothesis that distinct networks of 3D chromatin interactions are established throughout the somatotrope and lactotrope genomes to integrate and coordinate lineage-specific gene activation and repression programs and that these 3-D architectures are dependent on lineage-specific Pit-1 actions. All three Aims are based on analyses of primary cells isolated from the pituitaries of physiologically intact wild type or transgenic mouse lines. These studies will extend our understanding of pituitary function and will allow us to predict and define the basis for phenotypic variations in hormone expression, identify causative mutations in inherited and acquired endocrine disorders, and highlight targets for novel therapeutic interventions. Furthermore, this program will establish a paradigm of mammalian development in a landmark model and serve as a template for investigations of differentiation and genome regulation in a broad spectrum of experimental settings.